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 ~  Abstract
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 ~  Results and Disc...
 ~  Acknowledgement
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SPECIAL ARTICLE
Year : 2002  |  Volume : 20  |  Issue : 2  |  Page : 69-71
 

Rapid discrimination of Indian isolates of m.tuberculosis by random amplified polymorphic DNA (RAPD) analysis - A preliminary report


Dept. of Microbiology and Molecular Biology Central Jalma Institute of Leprosy (ICMR), Tajganj, Agra 282 001, India

Correspondence Address:
Dept. of Microbiology and Molecular Biology Central Jalma Institute of Leprosy (ICMR), Tajganj, Agra 282 001, India

 ~ Abstract 

PURPOSE: Due to relatively complex nature of molecular typing systems for M. tuberculosis as well as lack of applicability of some of the probes, there is a need for alternate procedures for molecular epidemiology. In this study the usefulness of RAPD analysis for typing of Indian strains of M.tuberculosis was investigated. METHODS: One hundred and three coded isolates from different parts of the country were analysed by Random amplified polymorphic DNA (RAPD) technique. Purified and amplified DNA from cultures were analysed by ethidium bromide staining after electrophoresis. The bands were confirmed by densitometry and the patterns were analysed by hierarchical cluster analysis. RESULTS: The patterns elicited by the analysis appeared to be quite discriminatory and characteristic. CONCLUSIONS: Clustering observed among isolates attending the same hospital indicates future application potential of RAPD analysis for molecular epidemiology of tuberculosis in India.

How to cite this article:
Singh H B, Chauhan D S, Singh D, Das R, Srivastava K, Yadav V S, Kumar A, Katoch V M, Sharma V D. Rapid discrimination of Indian isolates of m.tuberculosis by random amplified polymorphic DNA (RAPD) analysis - A preliminary report. Indian J Med Microbiol 2002;20:69-71


How to cite this URL:
Singh H B, Chauhan D S, Singh D, Das R, Srivastava K, Yadav V S, Kumar A, Katoch V M, Sharma V D. Rapid discrimination of Indian isolates of m.tuberculosis by random amplified polymorphic DNA (RAPD) analysis - A preliminary report. Indian J Med Microbiol [serial online] 2002 [cited 2014 Oct 1];20:69-71. Available from: http://www.ijmm.org/text.asp?2002/20/2/69/8349


Tuberculosis has re-emerged as one of the leading causes of death all over the world causing nearly three million deaths annually.[1] Investigations in the epidemiology of tuberculosis need strain specific markers, which can be used to differentiate M. tuberculosis isolates.[2] Restriction fragment length polymorphism (RFLP) using probes for insertion sequence IS986 or IS6110 has been extensively used to differentiate strains of M. tuberculosis.[2] However, the relatively complex nature of the methods, and lack of applicability of some of the probes such as IS 6110 to a section of Indian strain2,3 indicate the need for alternate rapid procedures. Random amplified polymorphic DNA (RAPD) also referred to as arbitrary primer PCR, is a DNA finger printing method which is easy to use, rapid and needs one day against 3-4 days required for other RFLP methods. RAPD has successfully been used for the differentiation of M. tuberculosis strains.4-6 In this report, the usefulness of RAPD analysis using an arbitrary primer 986 FP4 for the typing of Indian strains of M. tuberculosis has been investigated.

 ~ Materials and Methods Top

In this study 103 coded isolates of M. tuberculosis deposited in the Mycobacterial Repository Centre of our Institute from different areas of the country during 1999 (40 New Delhi Tuberculosis Centre; 19 MH Ranchi; 16 PGI Chandigarh; 7 IMS Varanasi; others from Institutes in Ahmedabad, Lucknow; Chennai, Agra and Sholapur) have been analysed by RAPD. For this analysis the isolates were grown on Lowenstein Jenson slants and DNA were purified by treatment with Lysozyme/proteinase K.[7] Amplification was done in a thermal cycler (model PTC-100, MJ Research, USA) by using the procedure described earlier4 (primer 986, 5 'ACG CTC AAC GCC AGA GAC CA 3', HPLC grade; Rama Biotechnologies Pvt. Ltd. India) The gene amplification products were analyzed by ethidium bromide staining after electrophoresis in a gel containing 2% agarose. Gels were photographed, and band patterns were compared visually and confirmed by densitometry (Gene Genius, UK) Patterns of bands generated were compared by using hierarchical cluster analysis (SPSS Software, SPSS Inc. USA) for the analysis of relationships.

 ~ Results and Discussion Top

RAPD profiles from all of the isolates included in the study were found to be consistent, discriminatory [Figure - 1] and reproducible as seen by patterns elicited by amplifying the same DNA on two different days and electrophoresing together. Relationships were investigated by arbitrarily classifying the isolates into 30 clusters. As seen in [Figure:2], while there were no clear patterns of relationships to geographical area in most of the clusters, there was clustering of isolates from same hospital isolated from different patients on different occasions (VK series from Military Hospital, Ranchi; ND/NDT from New Delhi Tuberculosis Centre; CHN from Chandigarh).
While no tracing of sources (contacts) in a given area was contemplated in this study, clusters among isolates collected from same source from different patients may indicate some transmission in those patients who may have close contact in the community they belong to. This RAPD system was earlier reported to be discriminatory in isolates from Britain, Gambia, Tibetan refugees in India, Malawi, Kenya and Saudi Arabia,[4] Taiwan[5] and South Africa.[6] Even though other arbitrary primers need to be analysed and protocols/ manuals are yet to be developed for other RAPD systems, our preliminary results indicate clear potential for future application of this technology for molecular epidemiology of tuberculosis in India. It would be important to take into consideration the factors possibly responsible for variation such as purity of primers, quality of DNA, standardized electrophoresis protocol and scanning. It would be important to investigate this strategy in well planned prospective studies and also compare with other methods for determining its ultimate application in India.

 ~ Acknowledgement Top

Authors are grateful to all contributors of isolates to Mycobacterial Repositary Centre especially Dr M.Hanif, New Delhi Tuberculosis Centre; Dr V. K. Kataria, Military Hospital Ranchi; and Dr Meera Sharma, PGI, Chandigarh. Gift of reagents/plasticware by LEPRA (UK) is gratefully acknowledged. This study was supported by a grant from Department of Biotechnology, Govt. of India (Grant No. BT/ 03/ 04 /94- PID). 

 ~ References Top

1.Bloom BR, Murray CJL.Tuberculosis: Commentary on a re-emergent killer, Science 1992; 257:1055-1064.  Back to cited text no. 1    
2.Katoch VM, Singh D, Chauhan DS, Sharma VD, Singh HB, Das R, Srivastava K. Newer DNA fingerprinting techniques for tuberculosis- relevance in control. In: Multi- drug resistance in emerging and re-emerging diseases , Mahajan RC, Therwath A ,Eds (Indian Science Academy and Narosa Publishing House, New Delhi) 2000: 87-96.  Back to cited text no. 2    
3.Das S, Paramasivan CN, Lowerie DB, Prabhakar R, Narayanan PR. IS 6110 Restriction fragment length polymorphism typing of clinical isolates of Mycobacterium tuberculosis from patients with pulmonary tuberculosis in Madras, India. Tuber Lung Dis 1995; 76: 550-554.  Back to cited text no. 3    
4.Linton CJ, Jalal H, Leeming JP, Millar MR. Rapid discrimination of Mycobacterium tuberculosis strains by random amplified polymorphic DNA analysis. J Clin Microbiol 1994; 32: 2169-2174.  Back to cited text no. 4    
5.Harn HJ, Shen KL, Ho LI, Yu KW, Liu GC, Yueh KC, Lee JH. Evidence of transmission of Mycobacterium tuberculosis by random amplified polymorphic DNA (RAPD) fingerprinting in Taipei city, Taiwan. J Clin Pathol 1997; 50: 505-508.  Back to cited text no. 5    
6.Richner SM, Meiring J, Kirby R. A study of genetic diversity of Mycobacterium tuberculosis isolates from patients in the eastern province of South Africa using random amplified polymorphic DNA profiling. Electrophoresis 1997; 18: 1570-1576.  Back to cited text no. 6    
7.van Embden JDA, Cave MD, Crawford JT, Dale J, Eisenach KD, Gicquel B, Hermans P, MartinC, Medam R, Shinnick TM, Small PM. Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: Recommendations for standardised methodology. J Clin Microbiol 1993; 31: 406-409.   Back to cited text no. 7    
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